When the vehicle is in motion, the elastic deformation of the flexible subframe significantly influences ride comfort. Therefore, it is crucial to investigate the impact of flexible subframes on vehicle ride comfort. In order to enhance the reliability and optimization efficiency of our research, this paper incorporates the concept of elastic deformation in the flexible subframe into the investigation of vehicle ride comfort, and proposes a multi-objective optimization approach to enhance the overall vehicle ride comfort. The vibration mathematical model elucidates how flexible subframes affect vehicle ride comfort and establishes a rigid-flexible coupling model for a specific vehicle with a flexible subframe to analyze the impact of its elastic deformation on vehicle ride comfort through simulation experiments. Subsequently, a radial basis function approximation model is established, and the multi-objective particle swarm optimization and non-dominated sorting genetic algorithm II algorithms are employed to conduct multi-objective optimization of the stiffness of the subframe bushing with the aim of enhancing vehicle ride comfort. The findings indicate that the flexible subframe has a significant impact on vehicle ride comfort. Specifically, on bump roads, peak values of vertical and longitudinal seat accelerations decrease while lateral seat acceleration increases. On random roads, peak values of longitudinal and lateral seat accelerations increase while vertical acceleration decreases. Furthermore, the stiffness of the subframe bushing optimized by the non-dominated sorting genetic algorithm II algorithm further enhances vehicle ride comfort and aligns more closely with the optimization requirements in this study.